2. Materials and Methods
2.5. Immunofluorescence techniques
2.5.6. Immuofluorescence for lamp1/2 and DAPI staining
After fixation, either with methanol or PFA, the cells were washed twice with PBS and blocked with 50mM NH4Cl solution in PBS for 10 min. Then cells were permeabilized with 0,05% Triton X-100 in PBS 2 times 5 minutes each and blocked with 1% BSA in PBS solution for 20 minutes. Following this, the cells were stained with primary antibody. The primary anti lamp1/2 antibody was diluted 1:100 in 1% BSA PBS solution. 20 µl of the antibody solution was pipetted onto a parafilm. Coverslips were placed with cell side down on the drop of antibody solution in a dark humid chamber. Cells were incubated with the antibodies for 1h in a humid dark chamber. After incubation, the coverslips were placed back into the wells with the cell side up. The cells were washed 3 times for 5 minutes each with 1x PBS and incubated with 10% goat serum in PBS for 20 min. Secondary antibody anti-rat Cy3 was diluted 1:200 in 10% goat serum solution and 20 µl was pipetted on fresh parafilm. The cells were again placed upside down on the drop of antibody solution for 45 minutes at room temperature in a dark humid chamber. Cells were then washed 5 times for 5 minutes each with 1xPBS and rinsed twice times with water. The coverslips mounted in DAKO mounting medium containing DAPI stain (DAKO®, Carpinteria, CA) and allowed to dry overnight at room temperature.
Chapter 2. Materials and methods
Table X: List of antibodies used for FACS analysis
Surface staining of T-lymphocytes
Antibody Conjugate Concentration Source Marker for Primary antibodies
Anti Mouse CD 4 PE 50µg/500µl Caltag T lymphocyte
Anti Mouse CD 4 TC 100µg/500µl Caltag T lymphocyte Anti Mouse CD8a FITC 100µg/1ml Caltag T lymphocyte
Anti Mouse CD8a PE 50µg/500µl Caltag T lymphocyte
Anti Mouse CD8b PE 50µg/500µl Caltag T lymphocyte
Anti Mouse CD3 PE 50µg/500µl Caltag T lymphocyte
Anti Mouse CD25 FITC 0.5 mg/ml BD
Pharmingen Immature thymocyte
Anti Mouse CD44 Biotin 100µg/1.0ml Caltag Immature thymocyte
Anti Mouse B220 PE 50µg/500µl Caltag B lymphocyte
Anti Mouse DX5 PE 0.5 mg/ml BD
Pharmingen Natural killer cells Anti Mouse MHCI FITC 100µg/1.0ml Caltag MHC I+ cells Anti Mouse TCRαβ FITC 100µg/1.0ml Caltag TCRαβ+mature T-
lymyphocytes
Anti Mouse Annexin V FITC 50µg/500µl BD
Pharmingen Apoptotic cells Anti Mouse CD19 FITC 100µg/1.0ml Caltag B lymphocyte Anti Mouse NK1.1 FITC 100µg/1.0ml Caltag Natural killer cells Anti Mouse MHCII FITC 100µg/1.0ml Caltag MHCII+dendritic cells
Isotype controls Control for
Anti Rat IgG2a PE 50µg/500µl Caltag CD8a (FITC and PE), CD4 (PE and TC), B220 (PE), CD3(PE)
Anti Rat IgG2b Biotin 50µg/500µl Caltag CD44 (Biotin)
Anti Rat IgM FITC 50µg/500µl Caltag CD25(FITC),DX5 (PE)
Anti Mouse IgG2a PE 50µg/500µl Caltag NK1.1 (PE)
Anti Mouse IgM FITC 50µg/500µl Caltag MHCII (FITC)
Hamster anti Mouse IgG FITC 50µg/500µl Caltag TCRαβ (FITC) Secondary antibody for Biotin conjugated anti-CD44 antibody and its isotype control rIgG2b
Streptavidin TC 50µg/500µl Caltag
Chapter 2. Materials and methods
Table XI: List of antibodies used for apoptosis induction assays
Plate bound CD3ε and CD28 induced apoptosis assay
Concentration
(37.51) 500µg/ml 10µg/ml BD Pharmingen
Anti FAS antibody induced apoptosis assay
Antibody Concentration Source
Purified anti mouse Fas monoclonal Clone Jo2 (Cat no 554254)
1.0mg/ml BD Pharmingen
Table XII: List of antibodies used for western blot analysis and immuno fluorescence Antibodies against SNARE proteins for western blotting
Protein Name Mol.
weight
Type Dilution Source
Vti1a 56 27 Polyclonal
(Rabbit serum) 1:3000 Antonin et al. 2000c Vti1b 55 29 Polyclonal
(Rabbit serum) 1:3000 Antonin et al. 2000c Endobrevin
/VAMP8 16 Polyclonal
(Rabbit serum) 1:1000 Synaptic Systems GmBH, Göttingen
Syntaxin 6 31 Monoclonal
(Mouse) 1:5000 Transduction Laboratories.
Syntaxin 8 60 27 Polyclonal
(Rabbit serum) 1:1000 Antonin et al. 2000b Syntaxin 7 Sx7/8 30 Polyclonal
(Rabbit serum)
1:1000 Antonin et al. 2000b SNAP 29 Sn29/1 29 Polyclonal
(Rabbit serum) 1:1000 Antonin et al. 2000b
Syntaxin16 ODTH 37 Polyclonal
(Rabbit serum)
1:1000 Kreykenbohm et al. 2002 VAMP4 136 19 Polyclonal
(Rabbit serum) 1:1000 Kreykenbohm et al. 2002
Antibodies for immunofluorescence
Antibody Dilution Source
Lamp1 (Rat monoclonal ID4B) 1:200 Hybridoma Bank, Iowa, USA Lamp2 (Rat monoclonal ABL93) 1:200 Hybridoma Bank, Iowa, USA
Anti rat - Cy3 1:200 Dianova
Chapter 2. Materials and methods
List of commonly used buffers
Buffer Composition
NET Buffer 100mM NaCl, 10mM Tris/HCl pH8.0, 25mM EDTA
TBS 150mM NaCl, 50mM Tris/HCl pH7.4
TE 1mM EDTA, 10mM Tris/HCl pH 7.4
10x PBS 1.5M NaCl, 160mMNa2HPO4, 40mM NaH2PO4
1M Tris/HCl (1000ml) 121g Tris base dissolved in 800 ml bidistilled H2O, pH adjusted with HCl and volume was made up till 1000 ml
50x TAE 2M Tris-base, 0.1M EDTA, adjust pH 8,0 with acetic acid 1x TAE 50x stock solution dissolved 1:50 in VS water
List of media used for cell culture
Dulbeccos Modified Eagle Medium - DMEM
Component Source Volume/Concentration
DMEM PAN biotech, GmBH 1000ml
Fetal calf serum Gibco BRL, Eggenstein 10%
L-glutamine Gibco BRL, Eggenstein 2mM Penicillin/streptomycine Gibco BRL, Eggenstein 100 U/ml Mix the components and pre warm the medium before use
Starvation Medium
Component Source Volume/Concentration
DMEM PAN biotech, GmBH 1000ml
L-glutamine Gibco BRL, Eggenstein 2mM Penicillin/streptomycin Gibco BRL, Eggenstein 100 U/ml Mix the components and pre warm the medium before use
Medium for cryoconservation of cells
Component Volume (for 10ml medium)
DMEM complete (ref table ) 7 ml
Fetal calf serum 2 ml
DMSO 1 ml
1ml of cryo preservation medium for each cryo tube.
Chapter 2. Materials and methods
Roswell Park Memorial Institute medium - RPMI 1640
Component Source Volume/Concentration
RPMI 1640 Gibco BRL, Eggenstein 1000ml Fetal calf serum Gibco BRL, Eggenstein 10%
L-glutamine Gibco BRL, Eggenstein 2mM Penicillin/streptomycin Gibco BRL, Eggenstein 100 U/ml Mix the components and pre warm the medium before use
Chapter 3. Results
3. Results
3.1 Ablation of VAMP8/endobrevin causes partial early mortality 3.1.1. Progressively degenerating health and early death in VAMP8-/- mice
The birth and death of VAMP8-/- Vti1b-/-, VAMP8-/- Vti1b+/-, VAMP8-/- and VAMP8+/ -Vti1b-/- pups was recorded. It was seen that all the VAMP8-/- genotypes had a high rate of mortality before the age of one month. During the initial work (Ref. section 1.4.4) 30 out of 45 (66%) VAMP8-/- Vti1b-/- and 20 out of 34 (58%) VAMP8-/- Vti1b+/- were reported dead before the age of one month (Figure 3.1). In this work, similar recording of births and death for VAMP8-/- showed that 20 out of 55 i.e. 36% VAMP8-/- mice were dead before one month of age (Figure 3.1). In comparison only 2.2% deaths were observed in VAMP8+/ -Vti1b-/- mice (Figure 3.1).
0 20 40 60 80 100
Number of dead mice
Total animals Dead before 1 mth Older than 1mth
Total animals 45 34 88 55
Dead before 1 mth 30 20 2 20
Older than 1mth 15 14 86 35
VAMP8-/- Vti1b-/- VAMP8-/- Vti1b+/- VAMP8+/- Vti1b-/- VAMP8-/-
Figure 3.1. High early mortality in VAMP8/endobrevin-/- genotypes: There was a high early mortality in VAMP8-/- mice compared to the VAMP8+/- mice. While 30 out of 45 VAMP8-/- Vti1b-/- and 20 out of 34 VAMP8 -/- Vti1b-/+ mice died before reaching the age of 1 month. The VAMP8 single knock out mice had an early onset of death and 20 out of 55 mice died before the age of weaning. VAMP-/+ mice however were more like the wild type mice with a very low rate of mortality (less than 2%).
VAMP8-/- and VAMP8-/-Vti1b-/- mice were born in expected Mendelian ratios indicating a normal pre-natal development. The mice also showed a normal post natal development up to day 7. After the 8th day, 36% of the VAMP8-/- mice began to loose weight and became smaller than the littermates. Loss of weight was observed till up to 2-3 days accompanied by progressively degenerating health, following which these mice died (Figure 3.2).
Chapter 3. Results VAMP 8 Heterozygous 1 VAMP 8 Heterozygous 2
Small not sick Small and sick
Figure 3.2. Weight gain pattern in VAMP8-/- mice: Characteristic pattern of weight gain of a representative litter shows heterogeneity in the phenotype of VAMP8-/- mice. One out of the two VAMP8 -/- pups lost weight starting post natal day 8 for 2-3 consecutive days, became sick and eventually died.
While the other VAMP8-/- pup survived the weaning and became adults. At the first day of weight loss, the mice were smaller than their littermates but were apparently active and did not look sick. This stage is called small not sick. While after 2-3 days of weight loss, the mice looked very weak and sick and this stage is described as small and sick.
After the first day of weigh loss, the mice were smaller than their littermates by around 1.5 folds. weighing 3.6 grams on the day of death compared to a VAMP8+/- littermate (left) weighing 6.3 grams on the same day (A). The average weight of small and sick mice is reduced at an average by 1.9 folds (n=7, age 8-12 days, left graph) while the mice at the preceding small not sick were lighter by 1.5 folds (n=4, age 8-12 days, right graph) compared to littermate controls (B).
Chapter 3. Results
Following this, there was a progressive decrease in weight such that at the time of death, the VAMP8-/- small and sick mice weighed almost half (average 1.9 fold decrease) that of the heterozygous littermates (Figure 3.3 A, B). The mice were very weak with a shivery and unstable gait
Similar pattern of weight loss and early mortality was previously observed in the VAMP8-/ -Vti1b-/- and VAMP8-/-Vti1b-/+ (Ref. section 1.4.4). However in contrast to VAMP8-/- mice that had a much early and sudden onset of the phenotypic manifestation and most of the VAMP8-/- Vti1b-/- mice died between post natal day 15-30. At the end of one month, just
Record of birth and death of VAMP8-/- Vti1b-/- double knock out and VAMP8-/ -Vti1b+/- mice. The VAMP8-/ -Vti1b-/- knock out mice showed the highest number of deaths with just about 33% surviving mice at the end of one month.
The VAMP8-/- Vti1b -/+ mice also had a high death rate with about 41% survivors after one month.
The VAMP8-/- and VAMP8-/- Vti1b-/- mice that survived past the initial one month of post natal life, reached adulthood and were fertile. However, they were in most cases lighter and smaller than their heterozygous littermates.